The research program described here is concerned with fundamental aspects of gene regulation by the steroid hormone ecdysone. Ecdysone regulates the development of insects and induces metamorphosis. The functional ecdysone receptor is a heterodimer of the proteins EcR and USP; both of these polypeptides are members of the nuclear hormone receptor family. EcR is most closely related to thyroid, retinoic acid, and vitamin D receptors and USP to the receptor RXR. The EcR/USP heterodimer interacts with an ecdysone response element (EcRE). In the absence of hormone this interaction inhibits transcription; in the hormone's presence it stimulates The major emphasis of this proposal is on analysis of the interactions between the two proteins EcR and USP, the hormone ecdysone, a putative ligand for USP, and the DNA binding site. Among EcREs which have been identified in ecdysone-responsive genes, there is no obvious relationship between their affinity for EcR/USP in vitro, their ability to mediate ecdysone induction in the Drosophila Kc cell line, and their ability to inhibit basal expression. Furthermore, particular EcREs display tissue- specific function. A variety of biochemical techniques will be used to understand how the sequence of an EcRE alters the activity of the receptor complex bound to it. The ligand for USP is unknown, but recent work in our laboratory suggests that it may play an important role in stimulating EcR/USP DNA binding, a hypothesis that will be tested further. Other experiments are proposed to express functional fragments of EcR and USP, to study their functions, to reveal the roles of various domains of these proteins, and to prepare the reagents necessary for detailed structural studies. EcR homologs diverge in sequence rapidly and distant homologs will be recovered for comparative structural and functional analysis. We discovered a novel form of gene targeting that occurs in Drosophila cells and we plan to use it to create EcR-deficient and USP-deficient cell lines; these will be important assets in future studies of ecdysone regulation. Finally, our work on the ecdysone-responsive gene Eip71CD has revealed a role for a transcription factor that may be a homolog of the vertebrate lymphocyte-specific transcription factor LyF-1/lkaros. We plan to clone and study this transcription factor.